Objective: To delineate the dynamic 3D genome reprogramming patterns during differentiation of normal human primary airway epithelial stem cells, uncover the critical chromatin switches driving the fate bifurcation into ciliated and goblet cells, and identify 3D structural abnormalities in basal cells in COPD, thereby establishing a paradigm from "map drawing" to "mechanism elucidation."

 

Methods: An air-liquid interface (ALI) differentiation model was used, and cells were collected on days 0 and 21. Multi-omic profiling was performed using Hi-C, CUT&Tag (H3K27ac, CTCF), and RNA-seq to characterize dynamics of compartments, TAD boundaries, and chromatin loops. For candidate loops and TADs, CRISPR-dCas9 was employed to artificially construct loops and TADs in basal cells, followed by observation of differentiation phenotypes. Additionally, a COPD mouse model was used to validate 3D structural alterations in basal cells.

 

Results: During normal differentiation, distinct cell types exhibited multi-level 3D genomic differences (loop strength, TAD boundaries, compartment switching, and chromosome-level changes). Multi-omic analysis revealed extensive 3D genome reprogramming during the transition from basal cells to ALI-BC, characterized by a genome-wide switch from B to A compartments, indicating that early differentiation is a critical window for 3D structural reorganization. Terminally differentiated goblet and ciliated cells displayed cell type-specific chromatin loops, with goblet cells showing more pronounced loop changes that drive their unique transcriptional programs (e.g., mucus secretion). CTCF-mediated loops and H3K27ac analysis further elucidated the regulatory mechanisms of loop formation. TAD reorganization (including boundary alterations and changes in internal interactions) was closely associated with ciliated cell differentiation and functional features, with novel TADs emerging at specific sites. Stable TAD boundaries were enriched for CTCF binding sites, whereas dynamic boundaries were not, confirming the essential role of CTCF in maintaining boundary stability. Compared with normal mice, COPD mice showed weakened 3D genomic interactions in airway epithelial basal cells. Although A/B compartment switching was minimal (<5%), Hi-C revealed widespread loss of interactions, with significant chromatin loops present in normal mice being absent in COPD mice.

 

Conclusion: This study presents the first dynamic 3D genome map of airway epithelial differentiation, demonstrating that temporal reprogramming of chromatin spatial conformation is a core mechanism driving the fate determination of ciliated and goblet cells. The weakened 3D architecture of basal cells in COPD may represent a critical source of aberrant epithelial remodeling. Integrating multi-omics with CRISPR validation achieves a transition from correlation to causation, providing a new theoretical paradigm for understanding airway epithelial homeostasis, remodeling, and COPD pathogenesis.

 

Keywords: dynamic 3D genome reprogramming; airway epithelial lineage differentiation; chromatin loop; multi-omics integration; chronic obstructive pulmonary disease